The euactinomycetes are a subset of a large and complex group of Gram-positive bacteria known as actinomycetes. Over the past few decades these organisms, which are abundant in soil, have generated significant commercial and scientific interest as a result of the large number of therapeutically useful compounds produced as secondary metabolites. The intensive search for strains able to produce new secondary metabolites having potential therapeutic applications has led to the identification of hundreds of new species. Many of the euactinomycetes, particularly Streptomyces and the closely related Saccharopolyspora genera, have been extensively studied. Both of these genera produce a notable diversity of biologically active metabolites. Because of the commercial significance of these compounds, much is known about the genetics and physiology of these organisms.
Microbial genomic information is unique in that, unlike the organization of genomic information in higher life forms, microbial secondary metabolic biosynthetic genes are known to cluster together within the genome. This information allows identification of the gene locus encoding the enzymes responsible for the biosynthesis of a specific molecule. Equally, the identification of the genes present within a cluster allows prediction of the structure of the secondary metabolite. The identification of the genes and proteins responsible for the production of active molecules allows for example, generation of structural analogs or improvement of the production process.
U.S. patent application Ser. No. 10/762,107 describes a dibenzodiazepinone secondary metabolite, specifically 10-farnesyl-4,6,8-trihydroxy-dibenzodiazepin-11-one (named ECO-04601) produced by a known euactinomycetes strain, Micromonospora sp. (IDAC 231203-01). Likewise, U.S. Pat. No. 5,541,181 (Ohkuma et al.) also discloses a dibenzodiazepinone secondary metabolite, specifically 5-farnesyl-4,7,9-trihydroxy-dibenzodiazepin-11-one (named “BU-4664L”), produced by a known euactinomycetes strain, Micromonospora sp. M990-6 (ATCC 55378). Both these dibenzodiazepinones have been reported to have anti-tumor activity.
Although many biologically active compounds have been identified from bacteria, there remains the need to obtain novel naturally occurring compounds with enhanced properties. Current methods of obtaining such compounds include screening of natural isolates and chemical modification of existing compounds, both of which are costly and time consuming. Current screening methods are based on general biological properties of the compound, which require prior knowledge of the structure of the molecules. Methods for chemically modifying known active compounds exist, but still suffer from practical limitations as to the type of compounds obtainable.
Thus, there exists a considerable need to obtain pharmaceutically active compounds in a cost-effective manner and with high yield. The present invention solves these problems by providing polynucleotides, polypeptides, vectors comprising the polynucleotides and host cells comprising the vectors for production of dibenzodiazepinones, as well as methods to generate farnesyl dibenzodiazepinones by de novo biosynthesis (heterologous or homologous expression of biosynthetic genes) or semi-synthesis rather than by chemical synthesis.